Methods for fermentative production of massoia lactone

ABSTRACT

The present invention relates to the field of fermentation biotechnology, more particularly to methods for the fermentative production of massoia lactone by Aureobasidium species.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is related to and claim priority to U.S.provisional patent application Ser. No. 62/205,996 filed 17 Aug. 2015.Each application is incorporated herein in its entirety.

SUBMISSION OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled2577249PCTSequenceListing.txt, created on 15 Aug. 2016 and is 294 kb insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to the field of fermentationbiotechnology, more particularly to methods for the fermentativeproduction of massoia lactone by Aureobasidium species.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

C-10 massoia lactone [C-10 (5,6-dihydro-6-pentyl-2H-pyran-2-one)] andC-12 massoia lactone [C-12 (5,6-dihydro-6-heptyl-2H-pyran-2-one)] arethe major constituent of bark oil of massoia (Cryptocarya massoia) [1].Massoia lactones can also be found in cane sugar molasses, cured tobaccoand the essential oil of Sweet Osmanthus (Osmanthus fragrans) andjasmine [2]. At 20 ppm, it has a taste described as creamy, coconut,green and slightly fruity. Massoia bark oil is used in the flavorindustry as an additive in butter and milk flavors (international FEMAcode 3744). Current global supply of massoia bark oil comes fromIndonesia, using primitive and costly extraction process that destroyprecious native forest.

Massoia lactone is the substrate for the production of saturateddelta-decanolide or delta-dodecanolide, which is the key molecule forpeach flavor. It can be made by biohydrogenation using a wide range ofmicroorganisms, e.g., yeast (Saccharomyces, Candida, Pichia), molds(Cladosporium), and bacteria (Pseudomonas, Sarcina), [3,4].

Although methods for the chemical synthesis of massoia lactone have beenavailable, the process is a rather complicated and requires multi-stepreactions using costly raw materials and catalysts [5]. Previously,massoia lactone was found in the alkali-hydrolyzed glycolipid secretedby Aureobasidium pullalan A-21 that was cultured under acalcium-deficient condition. It has been reported that, in the presenceof calcium, polymalic acid was produced instead [6]. The interactionbetween various trace elements on the production of massoia lactone hasnot been reported.

Certain strains of A. pullulans are known to produce extracellular“heavy oils”, or polyol lipids, when CaCO₃ is not present in the medium.The oils have been described as 3,5-dihydroxydecanoyl and5-hydroxy-2-decenoyl esters of arabitol and mannitol. Medium for oilproduction is composed of 50 g/l sucrose, 0.6 g/l (w/v) peptone, 0.4 g/lyeast extract, 5 g/l K₂HPO₄, 0.4% g/l MgSO₄*7H₂O, and 1 g/l NaCl [7].Another reported medium uses nitrate as the sole nitrogen source withlow phosphate content and is composed of 120 g/l glucose, 1.5 g/l NaNO₃,1 g/l KNO₃, 0.05 g/l KH₂PO₄, 0.2 g/l MgSO₄*7H₂O, 2 ppm ZnSO₄*7H₂O, and0.2 g/l yeast extract [6].

It is desired to develop new fermentation methods for the production ofmassoia lactone.

SUMMARY OF THE INVENTION

The present invention relates to the field of fermentationbiotechnology, more particularly to methods for the fermentativeproduction of massoia lactone by Aureobasidium species.

In one aspect, the present invention provides a method for thefermentative production of massoia lactone by Aureobasidium species. Inone embodiment, the Aureobasidium species is Aureobasidium melanogenum.In another embodiment, the A. melanogenum is a strain of A. melanogenumthat does not express a functional Aureobasidin A (AbA) biosynthesiscomplex (aba1) gene mRNA when cultured. In one embodiment, thefunctional mRNA is not expressed in the culture medium described herein.In a further embodiment, the A. melanogenum that does not express afunctional aba1 gene mRNA when cultured is the W5-2 strain of A.melanogenum as described herein. In one embodiment, the Aureobasidiumspecies described herein is cultured in a culture medium described infurther detail herein to produce a fermentation product containingmassoia lactone. In one embodiment, the culturing is performed for about4 days to about 12 days, preferably for about 5 days to about 12 days,more preferably for about 7 days to about 10 days. In anotherembodiment, the culturing is performed at about 25° C. to about 35° C.,preferably about 28° C. to about 32° C. In some embodiments, the massoialactone is purified from the fermentation product using conventionaltechniques and/or as described in further detail herein.

In a second aspect, the present invention provides a culture medium forthe fermentative production of massoia lactone. In one embodiment, theculture medium comprises high levels of phosphate ions, ammonium ionsand calcium ions as described in further detail herein. In someembodiments, the culture medium comprises KH₂PO₄, Na₂HPO₄, (NH4)₂SO₄,MgSO₄ and CaCl₂. In another embodiment, the culture medium furthercomprises at least two trace elements as described in further detailherein. In some embodiments, the trace elements may be selected fromFe²⁺, Cu²⁺, Zn²⁺ and MoO₄ ²⁻. In other embodiments, each trace elementthat may be present in the culture medium may be present in an amountfrom about 0.1 μM to about 1.0 mM. In a further embodiment, the culturemedium comprises urea as a nitrogen source. In another embodiment, theculture medium comprises glucose, mannose, xylose or mixtures thereof asa carbon source. In one embodiment, glucose is used as the sole carbonsource. In one embodiment, the culture medium has a pH from about 5.5 toabout 6.5, preferably about 6.0. The fermentation method produces highlevels of glycolipids. Biochemically, hydroxyl fatty acids conjugate toproduce sugars and eventually produce massoia lactone. Thus, the methodproduces high levels of massoia lactone—a commercially desirable featureof the present invention.

In a third aspect, the present invention provides a novel strain of A.melanogenum designated W5-2. In some embodiments, A. melanogenum W5-2does not express a functional Aureobasidin A biosynthesis complex genemRNA when cultured. In one embodiment, a functional mRNA is notexpressed in the culture medium described herein. In other embodiments,A. melanogenum W5-2 has been deposited with the Agricultural ResearchCulture Collection (NRRL), 1815 N. University Street, Peoria, Ill.61604, USA on 28 May 2015 and assigned accession number NRRL 67063.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a phylogenetic dendrogram, based on 18S rRNA gene, thefirst internal transcribed spacer (ITS1), the 5.8S rRNA gene, the secondITS region and the 5′ end of the 28S rRNA gene sequences and constructedfrom evolutionary distances, showing the position of Aureobasidiumstrain W5-2 within the radiation of members of the family Dothioraceae,order Dothideales [36]. Numbers at branching points refer to bootstrappercentages (based on 1000 resamplings); only values above 50% areshown. GenBank accession number of each sequence is shown inparentheses.

FIG. 2 shows results of a GPD1 CDS search by BLASTn against the WholeGenome Shortgun Motifs of Aureobasidium species. This search identifiedtwo highly related genomic sequences from Aureobasidium melanogenum CBS110374 scaffold_4_c3 and Aureobasidium pullulans AY4 contig20 (which isin fact A. melanogenum). Aureobasidium namibiae CBS, Aureobasidiumsubglaciale EXF-2481 and Aureobasidium pullulans EXF-150 had onlypartial sequences in the region. The phylogenic tree is generated BlastTree View at NCBI using fast minimal evolution method.

FIGS. 3A-3D show the characterization of Nile Red staining substance inW5-2 cells. Cell were cultured in HMDC medium with 10% glucose inshaking flasks for 5 day. Cells were stained with Nile Red and imaged byConfocal microscopy. FIG. 3A: Red channel. FIG. 3B: bright field image.FIG. 3C: Over-lay of FIG. 3A and FIG. 3B. Scale bar=10 μM. FIG. 3D: TLCimage of total ethyl acetate extract. The positions for triacylglyceride(TAG) and glycolipid are indicated on the right.

FIGS. 4A and 4B show GCMS analysis of Aureobasidium isolate W5-2. FIG.4A: GCMS spectra of methanol-esterified products of methanol andchloroform extract of freeze-dried cell biomass. Arrows indicate thepeaks for major fatty acid methyl esters and massoia lactone. FIG. 4B:Database search and the comparison of MS spectrum of the peak shown inupper panel to that of standard massoia lactone,6-Pentyl-5,6-dihydro-2H-pyran-2-one, shown in lower panel.

FIG. 5 shows using Central Composite Design (CCD) to optimizeconcentration of urea, trace elements and glucose.

FIG. 6 shows glycolipid profiles of various media. Medium composition isshown in Table 2. Seed culture was prepared by inoculating 1.5 ml offrozen 7% DMSO culture into 100 ml of the original HDCM in a 250 mlflask and cultured for 48 hrs at 30° C. The cells were harvested bycentrifugation, re-suspended in water and inoculated at 2% into eachmedium in 250 ml flasks. Cells were cultured at 30° C. with 200 rpmagitation. Samples shown are taken from the 6th day.

FIGS. 7A and 7B show the effects of trace elements. Media wereinoculated with water-washed cell stock cultured in HMDC medium to 0.1OD₆₀₀ and cultured at 30° C. with 200 rpm shaking for 8 days. FIG. 7A:Factorial Design was used to evaluate the effects of FeSO4, MnSO4,ZnSO4, CuCl2 and AlCl3. Run #6 (T2) and Run #12 (T3) were selected forfurther optimization. FIG. 7B: Factorial Design was used to evaluate theeffects of H3BO4, CoCl2 and NaMoO4 based on Medium 2 of step 5.

FIG. 8 shows production of massoia lactone with various carbon sources.W5-2 was cultured in T2 medium with glucose, mannose, xylose or glycerolas carbon sole source for 4 days. Each data set derived from 3biological replicates. Error bars indicates SD.

FIGS. 9A-9C show a comparison of massoia lactone yields in 4 media. Thesymbols for the media are shown in FIG. 9C. AM refers to a modifiedA-21M medium (MA-21M) containing 120 g/l glucose, 1.5 g/l NaNO₃, 1.0 g/lKNO₃, 0.05 g/l KH₂PO₄, 0.2 g/l MgSO₄, 0.0056 g/l FeSO₄, 0.2 g/l YeastExtract (pH 5.5). Seed culture was made in HMDC medium with completetrace elements. FIG. 9A: Massoia lactone. FIG. 9B: Dry biomass yield.FIG. 9C: Residual NH₄ ⁺ in the medium.

FIG. 10 shows a comparison of cells cultured in T0, T2 and T3 media.Cells were cultured in 2 L fermentor for 9 days.

FIG. 11 shows the effect of medium pH on massoia lactone yield. T2medium was adjusted to various pH from pH 4 to pH 6.5. Cells werecultured for 5 days.

FIGS. 12A-12C show production of massoia lactone. FIG. 12A: GCMSchromatograph of W5-2 Sample cultured in R15 Medium in shake flask. FIG.12B: GCMS chromatograph of W5-2 Sample cultured in T3 medium in 2 Lfermenter. FIG. 12C: database search of ML massoia lactone peak of (FIG.12B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the field of fermentationbiotechnology, more particularly to methods for the fermentativeproduction of massoia lactone by Aureobasidium species.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention belongs.

The term “about” or “approximately” means within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, morepreferably still within 10%, and even more preferably within 5% of agiven value or range. The allowable variation encompassed by the term“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.

The term “as described in further detail herein” means the descriptionof the embodiments set forth in the detailed description of theinvention and in the Examples. In this context, the Examples compriseadditional details of the general nature of the invention, as well ascertain specific aspects not found elsewhere in the specification.

The term “massoia lactone” as used herein means C-10 massoia lactone[C-10 (5,6-dihydro-6-pentyl-2H-pyran-2-one)] and/or C-12 massoia lactone[C-12 (5,6-dihydro-6-heptyl-2H-pyran-2-one)].

The term “Aureobasidium melanogenum W5-2” or “A. melanogenum W5-2” or“W5-2” refers to a novel strain of A. melanogenum isolated andcharacterized as described herein and a culture of which has beendeposited as described herein.

In one aspect, the present invention provides a method for thefermentative production of massoia lactone by Aureobasidium species. Inone embodiment, the Aureobasidium species is Aureobasidium melanogenum.In another embodiment, the A. melanogenum is a strain of A. melanogenumthat does not express a functional Aureobasidin A (AbA) biosynthesiscomplex (aba1) gene mRNA when cultured. In one embodiment, thefunctional mRNA is not expressed in the culture medium described herein.The sequence of a functional aba1 gene is set forth in Genbank AccessionNo. EU886741 (SEQ ID NO:5). In a further embodiment, the A. melanogenumthat does not express a functional aba1 gene mRNA when cultured is theW5-2 strain of A. melanogenum as described herein. In one embodiment,the Aureobasidium species described herein is cultured in a culturemedium described in further detail herein to produce a fermentationproduct containing massoia lactone. In one embodiment, the culturing isperformed for about 4 days to about 12 days, preferably for about 5 daysto about 12 days, preferably for about 5 days to about 11 days,preferably for about 6 days to about 11 days, more preferably for about7 days to about 10 days. In another embodiment, the culturing isperformed at about 25° C. to about 35° C., preferably at about 27° C. toabout 32° C., more preferably at about 28° C. to about 32° C. In afurther embodiment, the culturing is performed in shake flasks agitatedwith a speed from about 175 rpm to about 225 rpm, preferably about 200rpm. In some embodiments, the massoia lactone is purified from thefermentation product using conventional techniques and/or as describedin further detail herein.

In some embodiments, the massoia lactone is purified from thefermentation product using conventional techniques, e.g., by alkalinehydrolysis and solvent extraction [6]. In other embodiments, a stronginorganic acid is added to the fermentation product to hydrolyze thefermentation product. In one embodiment, the strong organic acid issulfuric acid or hydrochloric acid. In a further embodiment, the massoialactone is purified by solvent extraction or distillation. In someembodiments, the solvent is ethyl acetate or hexane.

In a second aspect, the present invention provides a culture medium forthe fermentative production of massoia lactone. In one embodiment, theculture medium comprises high levels of phosphate ions, ammonium ionsand calcium ions as described in further detail herein. In someembodiments, the culture medium comprises KH₂PO₄, Na₂HPO₄, (NH₄)₂SO₄,MgSO₄ and CaCl₂. In some embodiments, the culture medium comprises about10.0 g/l to about 15 g/l, preferably about 12.5 g/l KH₂PO₄, about 0.5g/l to about 2.0 g/l, preferably about 1.0 g/l Na₂HPO₄, about 3.5 g/l toabout 6.5 g/l, preferably about 5.0 g/l (NH₄)₂SO₄, about 1.0 g/l toabout 4.0 g/l, preferably about 2.5 g/l MgSO₄.7H₂O and about 0.10 g/l toabout 0.40 g/l, preferably about 0.25 g/l CaCl₂.2H₂O. In anotherembodiment, the culture medium further comprises at least two traceelements. In a further embodiment, the culture medium comprises at leastthree trace elements. In an additional embodiment, the culture mediumcomprises four trace elements. In some embodiments, the trace elementsmay be selected from Fe²⁺, Cu²⁺, Zn²⁺ and MoO₄ ²⁻. In other embodiments,each trace element that may be present in the culture medium may bepresent in an amount from about 0.1 μM to about 1.0 mM, from about 1.0μM to about 1.0 mM, from about 10.0 μM to about 1.0 mM, or from about100 μM to about 1.0 mM. In a further embodiment, the culture mediumcomprises urea or ammonium as a nitrogen source. In some embodiments,the urea is present in the culture medium in an amount from about 1.5g/l to about 2.5 g/l, preferably from about 1.8 g/l to about 2.2 g/l,more preferably about 2 g/l. In some embodiments, ammonium is present inthe culture medium in an amount to provide the same amount of nitrogenas provided by the noted urea amounts. In another embodiment, theculture medium comprises glucose, mannose, xylose or mixtures thereof asa carbon source. In some embodiments, the carbon source is present inthe culture medium in an amount from about 4% to about 12%, preferablyfrom about 5% to about 12%, preferably from about 5% to about 11%, morepreferably from about 5% to about 10%. In some embodiments, glucose isthe sole carbon source. In one embodiment, the culture medium has a pHfrom about 5.5 to about 6.5, preferably about 6.0.

In a third aspect, the present invention provides a novel strain of A.melanogenum designated W5-2, including a pure culture of the novelstrain or the isolated novel strain or the isolated and biologicallypure culture of the novel strain. In some embodiments, A. melanogenumW5-2 does not express a functional Aureobasidin A synthase gene mRNAwhen cultured. In one embodiment, the functional mRNA is not expressedin the culture medium described herein. In other embodiments, A.melanogenum W5-2 was deposited on 28 May 2015 under terms of theBudapest Treating with the Agricultural Research Culture Collection(NRRL) located at 1815 N. University Street, Peoria, Ill. 61604, USA andassigned Accession Number NRRL 67063.

In some embodiments, Aureobasidium melanogenum and/or Aureobasidiummelanogenum strain W5-2 is characterized by the sequence of its genome.In one embodiment, the Aureobasidium melanogenum GDP1 genomic sequenceshares at least 97.5% identity over at least 98.5% of SEQ ID NO:2,preferably 99%-100% identity to over at least 98% of SEQ ID NO:2. Inanother embodiment, the Aureobasidium melanogenum TEF1A genomic sequenceshares at least 98% identity over at least 94% of SEQ ID NO:8,preferably 99%-100% identity to over at least 99% of SEQ ID NO:8. In anadditional embodiment, the Aureobasidium melanogenum RBP1 genomicsequence shares at least 91% identity over at least 92% of SEQ ID NO:10,preferably 96%-100% identity to over at least 98% of SEQ ID NO:10. In afurther embodiment, the Aureobasidium melanogenum GDP1 genomic sequenceshares at least 97.5% identity over at least 98.5% of SEQ ID NO:2,preferably 99%-100% identity to over at least 98% of SEQ ID NO:2, theAureobasidium melanogenum TEF1A genomic sequence shares at least 98%identity over at least 94% of SEQ ID NO:8, preferably 99%-100% identityto over at least 99% of SEQ ID NO:8 and the Aureobasidium melanogenumRBP1 genomic sequence shares at least 91% identity over at least 92% ofSEQ ID NO:10, preferably 96%-100% identity to over at least 98% of SEQID NO:10.

In other embodiments, the Aureobasidium melanogenum and/or Aureobasidiummelanogenum strain W5-2 is characterized by the amount of fatty acidsstored in the fungal cells. In one embodiment, the Aureobasidiummelanogenum and/or Aureobasidium melanogenum strain W5-2 fungal cellscan store fatty acids at about 40% of its dry weight.

In accordance with the present invention, it has surprisingly been foundthat the fermentation of A. melanogenum W5-2 in the culture medium thatcontains all of the components described herein for the culture mediumproduces a very high yield of massoia lactone. For example, as shown inthe Examples, batch fermentation of A. melanogenum W5-2 in this culturemedium produced massoia lactone at a 7 day peak of 10.268 g/l in a 2 Lbioreactor, with a volume productivity of 61.11 mg/hr/l. Thus, in oneembodiment, the batch fermentation of A. melanogenum W5-2 in thisculture medium yields 10.268 g/l of crude massoia lactone. In anotherembodiment, the batch fermentation of A. melanogenum W5-2 in thisculture medium yields more than 10 g/l of crude massoia lactone. In someembodiments, the yield of crude massoia lactone is at least 11 g/l, orat least 12 g/l, or at least 13 g/l, or at least 14 g/l or at least 15g/l. In other embodiments, the yield of crude massoia lactone is fromabout 10 g/l to about 25 g/l, or from about 10 g/l to about 24 g/l, orfrom about 10 g/l to about 23 g/l, or from about 10 g/l to about 22 g/l,or from about 10 g/l to about 21 g/l, or from about 10 g/l to about 20g/l, or from about 10 g/l to about 19 g/l, or from about 10 g/l to about18 g/l, or from about 10 g/l to about 17 g/l, or from about 10 g/l toabout 16 g/l, or from about 10 g/l to about 15 g/l, or from about 10 g/lto about 14 g/l.

In some embodiments, the yield of purified massoia lactone from thecrude extract is at least 50%. In other embodiments, the yield ofpurified massoia lactone from the crude extract is more than 5 g/l. Insome embodiments, the yield of purified massoia lactone from the crudeextract is at least 5.5 g/l, or at least 6 g/l, or at least 6.5 g/l, orat least 7 g/l or at least 7.5 g/l. In other embodiments, the yield ofpurified massoia lactone from the crude extract from about 5 g/l toabout 12.5 g/l, or from about 5 g/l to about 12 g/l, or from about 5 g/lto about 11.5 g/l, or from about 5 g/l to about 11 g/l, or from about 5g/l to about 10.5 g/l, or from about 5 g/l to about 10 g/l, or fromabout 5 g/l to about 9.5 g/l, or from about 5 g/l to about 9 g/l, orfrom about 5 g/l to about 8.5 g/l, or from about 5 g/l to about 8 g/l,or from about 5 g/l to about 7.5 g/l, or from about 10 g/l to about 7g/l.

It has surprisingly been found that the fermentation method using strainW5-2, as described herein, has several advantages.

The method produces high levels of glycolipids. Biochemically hydroxylfatty acids conjugate to produce sugars and eventually produce massoialactone. Thus, the method produces high levels of massoia lactone—acommercially desirable feature of the invention.

Massoia lactone is exuded into the medium and about 80% of thesupernatant contains massoia lactone—reflecting the ability to obtainhigh amounts of massoia lactone.

The method can produce more than 10 g/l massoia lactone within 5 days atlab scale using glucose as a sole carbon source.

The crude extract has more than 10 g/l of massoia lactone. Uponpurification the yield is more than 5 g/l of the crude extract, which isat least about 50% distillation recovery.

Use of 2 liter bioreactor yields high amounts of massoia lactone.

The method is highly efficient—the crude extract has less of impurities,including negligible or minimum intermediates. The method is highly timeand cost efficient.

The massoia lactone is capable of producing multiple odors and/orflavors, e.g., coconut, waxy, oily aroma, creamy, green and slightlyfruity flavors and/or odors. The massoia lactone can be converted intodelta-decanolide or delta-dodecanolide to produce a peachy flavor and/orodor.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Russell, 1984, Molecular biology of plants: alaboratory course manual (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Anand, Techniques for the Analysis of ComplexGenomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide toYeast Genetics and Molecular Biology (Academic Press, New York, 1991);Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.); Nucleic Acid Hybridization (B. D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Fire et al.,RNA Interference Technology: From Basic Science to Drug Development,Cambridge University Press, Cambridge, 2005; Schepers, RNA Interferencein Practice, Wiley-VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts& Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference,Editing, and Modification: Methods and Protocols (Methods in MolecularBiology), Human Press, Totowa, N.J., 2004; Sohail, Gene Silencing by RNAInterference: Technology and Application, CRC, 2004.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 Materials and Methods

Isolation of Microbial Strains:

Microbial samples were collected from various regions in Singapore.Samples were mixed with 100 ml autoclaved sea water and incubatedovernight at 28° C. A 100 μl of the overnight culture was withdrawn andstreaked on modified seawater-YPD (Yeast Peptone Dextrose) medium(containing 1 g/l peptone, 2 g/l yeast extract, 4 g/l glucose, and 8 g/lagar, pH 7), which was further supplemented with 0.5 μg/ml Nile red andan antibiotic selected from ampicillin (25 μg/ml), kanamycin (25 μg/ml)and streptomycin (100 μg/ml). Potato dextrose agar (PDA) was also usedfor the initial screening. The plates were incubated at 28° C. for 48 hrand the strongly Nile red staining strains were identified by examiningthe plates under a Nikon C-DSS230 stereomicroscope microscope (Nikon,Japan) equipped with Digital Sight DS-L1 camera. Candidate strains werepurified by 2 rounds of single colony isolation on seawater-YPD platesfollowed by verification of Nile red staining in small scale liquidcultures. Florescence was measured using the TECAN infinite M200fluorometer with the excitation wave-length and emission wave-length setat of 530 nm and 575 nm respectively (TECAN, USA).

Phylogenetic Analysis of the Microbial Strains by ITS Sequencing:

Yeast isolates were cultured in YPD medium at 30° C. Genomic DNAs wereextracted from 2 ml of 48 hr cultures using the MasterPure™ Yeast DNAPurification Kit (Epicentre Biotechnologies, USA) according to themanufacturer's instructions. PCR amplification reactions were performedin 40 μl 1× buffer with 2.5 mM dNTP, 50 μM each primer, 50 ng of totalDNA and 3 units of Taq DNA polymerase (i-DNA Biotechnology, Singapore).PCR cycling conditions were as followed: 95° C./10 min, 30 cycles of 95°C./1 min, 61.8° C./1 min and 72° C./1 min and final extension for 5min/72° C. The ITS1(5′-tccgtaggtgaacctgcgg; SEQ ID NO:3) and ITS4(5′-tcctccgcttattgatatgc SEQ ID NO:4) [8] primers were used to amplifythe ITS region of the nuclear rRNA operon spanning the 3′ end of the 18SrRNA gene, the first internal transcribed spacer (ITS1), the 5.8S rRNAgene, the second ITS region and the 5′ end of the 28S rRNA gene. [9].Gel-purified PCR products were sequenced with the Big-Dye sequencingmethod in Applied Biosystems 3730×1 DNA Analyzer (Life technologies,USA). Sequences were analyzed by BLAST against the NCBI database andaligned using the CLUSTAL W tool in MEGA version 5.05 [10]. Phylogeneticanalyses were performed by the Neighbour-Joining [11],Maximum-Likelihood [12] and Maximum-Parsimony methods [13] using theMEGA version 5.05 with the bootstrap values set at 1000 replications.

Small Scale Culture and Fed-Batch Fermentation:

Aureobasidium strain W5-2 was cultured in 100 ml liquid medium in 250 mlshake flasks, agitated with a speed of 200 rpm and constant temperatureof 30° C. The High Density Culture Medium (HDCM) developed forRhodotorula glutinis [14] was used as the basic culture medium. Itcontains 90 g/l glucose, 12.5 g/l KH₂PO₄, 1.0 g/l Na₂HPO₄, 5.0 g/l(NH4)₂SO₄, 1.9 g/l yeast extract, 2.5 g/l MgSO₄.7H₂O, 0.25 g/lCaCl₂.2H₂O and 0.25 ml/l trace element mix (pH 5.5). Trace elements mixwas made in 5N HCl and contains 40 g/l FeSO₄.7H2O, 40 g/l CaCl₂.2H₂O, 10g/l MnSO₄.7H₂O, 10 g/l AlCl₃.6H₂O, 4 g/l CoCl₂, 2 g/l ZnSO₄.7H₂O, 2 g/lNa₂MoO₄, 1 g/l CuCl₂.2H₂O and 0.5 g/l H₃BO₃. Where indicated, levels oftrace elements and various nitrogen sources were varied. For comparisonpurpose, the strain was also cultured in the A-21M medium reportedpreviously [6], which contains 120 g/l glucose, 1.5 g/l NaNO₃, 1.0 g/lKNO₃, 0.05 g/l KH₂PO₄, 0.2 g/l MgSO₄.7H₂O, 2 ppm ZnSO₄, 0.2 g/l yeastextract (pH 5.5).

Fed-batch fermentation was carried out in a 2 L Biostat B plusbioreactor (Sartorius Stedim Biotech, Germany). Dissolved oxygen level(pO₂) and air flow was maintained at 30% and 1.5 vvm, respectively. 25ml samples were taken daily to monitor glucose, ammonium, NO²⁻ and NO³⁻levels.

Optimization of Massaio Lactone Production by Design of Experiment(DOE):

Optimization of massaio lactone production by Design of Experiment (DOE)was aided with the Design-Expert® V8 Software (Stat-Ease, USA) usingboth the Optimised Factorial Design and Central Composite Design (CCD).Seed cultures prepared in the HDCM medium were harvested bycentrifugation, washed with sterile distilled water and suspended insterile distilled water. A fraction of which (2 ml) was inoculated into100 ml of the respective designed medium in a 250 ml shake flask, whichwas cultured in a 30° C. shaking platform agitated at 200 rpm. Culturesamples (15 ml) were taken daily for the analyses of cell biomass, OD₆₀₀and metabolites.

Extraction and Quantification of Glycolipids:

Ethyl acetate was added to cell culture at a volumetric ratio of 1:1 ina 15 ml Falcon tube. The mixture was vortexed vigorously for 20 seconds;centrifuged at 3,500 g for 10 minutes and 1 ml of the upper phase wastransferred to a 1.5 ml Eppendorf tube and left to air dry overnight atroom temperature in an exhaust hood. The resultant dried residue wasweighed with a microbalance before being added with 10 μl menthol inmethanol (10% w/v) (as the internal standard) and 300 μl of 2M NaOH. Thesuspended mixture was allowed to hydrolyze overnight in a shakingplatform at room temperature. After mixing with 150 μl of 5M H₂SO₄, 450μl ethyl acetate was added and vortexed vigorously. Aftercentrifugation, the upper phase was used for GCMS analysis directly.Alternatively, glycolipids were extracted directly from wet cell biomasscollected by centrifugation from 10 ml culture. 2 ml of 72% H₂SO₄ wasadded to the cell pellet, mixed well and allowed to stand for half anhour and then mixed with 4 ml water. After boiling for one hour, 4 ml ofthe mixture was added with equal volume of ethyl acetate; vortexedvigorously for 20 seconds; centrifuged at 3,500 g for 10 minutes and 1ml of the upper phase was analyzed by GCMS or thin-layer chromatography(TLC). Conditions used TLC were as described previously [15]. Hexane canbe used in place of ethyl acetate in this procedure. HCl can be used inplace of H₂SO₄ in this procedure.

Preparation and Quantification of Massoia Lactone:

Equal volume of H₂SO₄ was added to 2 ml of cell culture in a 15 mlFalcon tube. Samples were vortexed briefly and left in room temperaturefor 30 mins. After adding 4 ml of water, Falcon tubes were boiled in awater-bath for 60 min; cooled for 10 mins at room temperature, and then4 ml of ethyl acetate was added into the tubes, which were mixedvigorously with a vortex for 20 seconds. Samples were centrifuged for 10mins at 3,500 ref and 1 ml of the top organic layer was transferred to a2 ml glass vial containing 50 mg of Na₂SO₄ and 10 μl menthol solution inmethanol (10%). Massoia lactone was quantified using GCMS using mentholas the internal standard. HCl can be used in place of H₂SO₄ in thisprocedure.

Quantification of Fatty Acid:

Cell biomass was collected from 20 ml culture by centrifugation anddried in a 60° C. oven until constant weight is reached. The resultantdried pellets were frozen in liquid nitrogen and grinded to a finepowder using mortar and pestle. A 250 mg sample was transferred to a 15ml Falcon tube and then mixed with 2 ml of 72% H₂SO₄ and 10 μl of 10%(w/v) pentadeconoic acid in methanol (as the internal standard). Thesamples were hydrolyzed at room temperature for 30 mins and then mixedwith 4 ml water. After boiling for 1 hour, a 500 μl fraction wastransferred to a 2 ml Eppendorf tube and fatty acids were extracted bymixing with 1 ml methanol/chloroform mixture (1:1 v/v). Aftercentrifugation, the bottom layer was collected by pipetting and washedonce with PBS buffer in an Eppendorf tube. The methanol/chloroformbottom layer was collected after centrifugation and left to dry at roomtemperature in an exhaust hood. 300 μl petroleum ester (FisherChemicals, CAS: 64742-49-0)/benzene (QREC Asia SDN BDH, CAS 71-43-2)mixture (1:1 v/v) and 300 μl 0.4 M KOH in methanol was added tosolubilize the dried residues. Esterification was performed at roomtemperature for 3 hours. After separation by centrifugation, 50 μl ofthe top layer was diluted with 450 μl of methanol and subjected toanalysis by GC-MS.

GC-Ms Analysis:

GC-MS analysis was performed using GCMS-QP2010 Ultra (ShimadzuCorporation, Japan). Samples (10) were injected into a HP-88 column (30m×0.25 mm ID×0.20 μm) (Agilent Technologies, USA) and run with helium asthe carrier gas maintained at 10 psi. A split-less injection time of 0.5min was used. The GC started at an initial temperature of 50° C. for 1minute, ramped at 15° C. per minute up to 150° C. and 3° C. per minuteto a final temperature 240° C. The spectrometer was scanned from 41-400amu. The compounds were identified by searching against the NIST 08 massspectral library. Quantification of fatty acid ester and massoia lactonewas done by comparing the peak area between the target compounds and therespective internal standards.

Other Quantification Methods:

Glucose levels were quantified using a Shimadzu Prominence UFLC(Shimadzu, Japan). Samples were run through an Aminex HPX-87H column(Bio-rad, USA) maintained at 50° C. 5 mM H₂SO₄ was used as the mobilephase and run at a flow rate of 0.7 ml/min. Total nitrates, nitrites andammonia levels were determined using the method as described [16, 17].

RNA Sampling and RNA-Sequencing:

Cell cultures (1 ml) were collected at day 1, 2, 3 and RNAs wereimmediately extracted with RiboPure™ RNA Purification Kit, yeast (LifeTechnologies, USA). After determination of the RNA quantity and qualityby Nanodrop and agarose gel electrophoresis, the RNA samples were sentto Macrogen Inc. (Korea) for cDNA shotgun library construction andsequencing using Illumina Hiseq 2000.

Computational Analysis:

Computational analysis was performed in the Galaxy platform(http://galaxyproject.org/) installed locally [18]. Raw reads (100 bppaired-end) were analyzed with NGS QC toolkit [19] for their quality.Then, Tophat and Cufflinks [20-22] were used to identify differentialgene/transcript expression based on published genome sequences as thereferences. de novo assembly was performed with Trinity [23, 24], whichproduced ˜18 k isoforms or transcripts. Differentially expressed (DE)transcripts and expression profile clusters were done with Bioconductorwith rsem and edgeR packages [25, 26].

Example 2 Identification of Massoia Lactone-Producing Microbial Strains

Nile red is an uncharged hydrophobic molecule whose fluorescence isstrongly influenced by the polarity of its environment and it is oftenused as a marker for hydrophobic substances, such as lipid, glycolipidsand hydrophobic proteins [27-29]. By screening water and soil samplesobtained from local costal environment using Nile red as a marker, 32candidates were identified that showed significant red fluorescence,among which strain W5-2 was identified as fungus closely related toAureobasidium species based on the sequence comparison of the rDNA ITSregion (SEQ ID NO. 1) (FIG. 1). W5-2 colonies turned black after 7 daysof culture on solid medium (not shown).

Because Aureobasidium species are identified by phylogenetic analysis oftheir whole genome sequences as well as certain phylogeneticallyimportant genes, such as house-keeping genes encoding Actin, β-tubulin,calmodulin, chytin synthase, NAD-dependent glycerol-3-phosphatedehydrogenase and translation elongation factor 1α (TEF1A), the wholetranscriptome of W5-2 was chosen to compare those of the typeAureobasidium strains [30].

The overall alignment rates of raw reads to the 4 reference genomes ofAurebasidium species [31] were low, ˜55.7% to A. melanogenum and ˜15-19%to the rest Aureobasidium species (Table 1). Therefore, de novo assemblyof RNA-seq data was performed using the Trinity program [32], producinga sequence library of ˜18 k isoforms/transcripts. The overall alignmentrate of raw reads to this local reference was increased to above 95%.

TABLE 1 Mapping Rates to Reference Genomes of Aurebasidium Species T0-1d T0-2 d T0-3 d T2-1 d T2-2 d T2-3 d Average A. melanogenum 60.47%52.76% 52.10% 58.24% 56.73% 54.06% 55.72% A. pullulans 21.68% 15.71%15.81% 16.80% 15.06% 13.86% 16.49% A. subglaciale 19.80% 14.23% 14.41%15.46% 13.48% 12.41% 14.97% A. namibiae 24.90% 18.11% 18.23% 20.08%18.04% 16.32% 19.28% Note: Raw reads were mapped to reference genomeusing TopHat software[33]. RNA samples were extracted from W5-2 cellculture in Medium T0 and Medium T2 at Day, 1, 2 and 3 respectively.

DNA sequence divergence in GPD1 gene is used as an important indicatorfor phylogenetic analysis in Aureobasidium species [30]. GPD1 CDSsequence (SEQ ID NO:2) was searched by BLASTn against the Whole GenomeShortgun sequences of Aureobasidium species at NCBI. Six highly relatedsequences were identified. Phylogenic tree generated Blast Tree Viewusing fast minimal evolution method is shown in FIG. 2, which clearlyplaces the W5-2 GPD1 sequence between A. melanogenum CBS 110374/A.pullulans AY4, which has been re-classified as A. melanogenum [30], andA. namibiae CBS 147.97/A. subglaciale EXF-2481. W5-2 showed highestdivergence from A. pullulans EXF-150 and lowest divergence to A.melanogenum.

A. pullulans is known to produce antibiotic aureobasidin A [34], whichis made by a huge polyketide synthase Aba1 [35]. A search of ABA1 CDS(Genbank no. EU886741) against the W5-2 whole transcriptome failed toidentify any homologs in the genome. Therefore, strain W5-2 does notencode a functional ABA1 gene and is not likely to produce anyantibiotics.

Confocal imaging confirmed that Nile red stained the cytoplasm intenselywith little signal in the cytoplasmic membrane, suggesting the highaccumulation of hydrophobic substance in the cells (FIG. 3C). Fatty acidprofiling by GCMS revealed that the total ethyl acetate extractcontained 52.28% oleic acid, 33.9% palmitic acid, 3.98% linoleic acid,1.31% palmatolic acid and about 1.3% of long chain (C24 and above) fattyacids (Table 2). Thin layer chromatography (TLC) confirmed theproduction of triacylglyceride (TAG) and glycolipids (FIG. 3D).Unexpectedly, the GCMS profile showed a significant peak with aretention time of approximately 25.7 min, which had >93% similarity tomassoia lactone, i.e., 6-pentyl-5,6-dihydro-2H-pyran-2-one (FIGS. 4A and4B). Sensory test of the dried cell pellet confirmed the presence astrong coconut-like aroma.

TABLE 2 Fatty Acid Profile of W5-2 C16 C16:1 C18 C18:1 C18:2 C24 W5-233.91% 1.31% 7.24% 52.28% 3.98% 1.29%

Example 3 Medium Optimization Using Central Composite Designs (CCD)

To improve massoia lactone production, the Rhodotorula glutinis highdensity (HMDC) medium [14] was modified by changing the levels ofnitrogen source (urea), carbon source (glucose) and trace element mix,which contains FeSO₄.7H₂O, CaCl₂.2H₂O, MnSO₄, AlCl₃.6H₂O, CoCl₂,ZnSO₄.7H₂O, Na₂MoO₄.2H₂O, CuCl₂.2H₂O and H₃BO₄. Medium compositions areshown in Table 3. Massoia lactone production was low, ranging from 0.05mg/l (Run 6) to 1.61 mg/l (Run 16) (FIG. 5). While there were obviousdifferences amongst the runs, none of the parameters appeared tosignificantly influence the production by ANOVA analysis (p<0.05) (Table4). Since massoia lactone produced in A. pullulans has been reported toderive from glycolipid [6], we monitored glycolipid levels by TLC.Again, CCD was employed to optimize the nitrogen source and carbonsource in HMDC medium, with urea and yeast extract set as categoryfactors while glucose level was set at 7.5, 20, 50, 80, 92.4 g/lrespectively. In addition, the trace element mix was set at 0.034, 0.2,0.6, 1.0, 1.17 ml per litre medium respectively (Table 5). Highvariations in glycolipid profiles were observed: Run No. 15 appeared tobe the best combination for glycolipid production (FIG. 6), with 285mg/l massoia lactone present in the acid hydrolyzed glycolipid products.Therefore, we chose a medium containing high level of glucose (˜100g/l), 2 g/l urea as the sole nitrogen source and 0.6 ml original HMDCtrace element mix for subsequent medium optimizations. For convenience,this medium is referred as the Run 15 (R15) medium.

TABLE 3 Optimization of Nitrogen source, Carbon Source and TraceElements by CCD Run No. Urea (g/L) Glucose (g/l) Trace (ml/l) 1 2.8936.21 0.95 2 1.11 83.78 0.24 3 2 60 1.2 4 2 60 0.6 5 0.5 60 0.6 6 2 60 07 2.89 36.21 0.24 8 2 60 0.6 9 2 60 0.6 10 2 60 0.6 11 3.5 60 0.6 121.11 83.78 0.957 13 1.11 36.21 0.96 14 2 20 0.6 15 2 60 0.6 16 2 60 1.0017 2.89 83.78 0.24 18 1.11 36.21 0.24 19 2 100 0.6 20 2.89 83.78 0.96

TABLE 4 Anova Analysis of Table 3 Sum of Mean F p-value Source Squaresdf Square Value Prob > F Remarks Model 0.75 3.00 0.25 1.98 0.1580 notsignificant A-Trace 0.23 1.00 0.23 1.86 0.1919 elements B-Urea 0.00 1.000.00 0.02 0.8813 C-Glucose 0.51 1.00 0.51 4.05 0.0612 Residual 2.0216.00 0.13 Lack of Fit 1.87 11.00 0.17 5.95 0.0308 significant PureError 0.14 5.00 0.03 Cor Total 2.77 19.00 Std. Dev. 0.36 R-Squared 0.27Mean 0.59 Adj R- 0.13 Squared C.V. % 60.42 Pred R- −0.30 Squared PRESS3.60 Adeq 4.27 Precision

TABLE 5 Medium Compositions of Central Composite Design 1 (CCD1)Nitrogen Glucose Trace element lactone Run source (g/l) mix (ml/l) titre(mg/l) 1 Urea 7.57 0.6 0.256 2 Yeast extract 50 0.03 4.192 3 Yeastextract 50 0.6 2.384 4 Yeast extract 7.57 0.6 1.386 5 Yeast extract 800.2 2.594 6 Yeast extract 20 0.2 3.257 7 Yeast extract 20 1 4.130 8 Urea80 0.2 3.082 9 Urea 50 1.17 4.978 10 Yeast extract 50 0.6 3.100 11 Yeastextract 80 1 3.366 12 Urea 20 1 1.853 13 Urea 20 0.2 4.103 14 Urea 80 14.935 15 Urea 92.43 0.6 6.731 16 Urea 50 0.6 2.300 17 Urea 50 0.6 5.27218 Urea 50 0.6 3.011 19 Yeast extract 50 0.6 4.325 20 Yeast extract 501.17 2.510 21 Yeast extract 50 0.6 2.651 22 Urea 50 0.6 5.715 23 Urea 500.03 4.990 24 Yeast extract 92.43 0.6 0.934 25 Urea 50 0.6 3.700 26Yeast extract 50 0.6 4.855 Note: urea and yeast extract were both set at1.9 g/l.

Example 4 Optimization of Trace Elements Using Factorial Designs

FeSO₄, MnSO₄, ZnSO₄, CuCl₂, AlCl₃ were supplemented to the R15 mediumusing Factorial Optimal Design at the same concentration [14], eitherindividually or in combinations (Table 6). Results showed that Run #12which was supplemented with Fe²⁺, Zn²⁺ and Cu²⁺ (named as T3 mediumhereafter) produced the highest level of massoia lactone, reaching 433mg/l on day 8. Run #6 in which Mn²⁺ and Zn²⁺ were both supplemented(named as T2 medium hereafter) ranked 2^(nd), yielding 218 mg/l massoialactone (FIG. 7A; Table 6). In contrast, Basal medium (Run 2, referredto as T0 medium) with CoCl₂, HBO₃ and NaMoO₄ only produced only 58 mg/lmassoia lactone, more than 14 folds lower than with T3 medium (FIG. 7A,Table 6). Anova analysis showed that ZnSO₄, CuCl₂, FeSO₄, ZnSO₄,FeSO₄—CuCl₂, FeSO₄—AlCl₃, and CuCl₂—AlCl₃ significantly affected massoialactone production (Tables 6 and 7).

TABLE 6 Trace Elements Part 1 Run # FeSO₄ MnSO₄ ZnSO₄ CuCl₂ AlCl₃ 1 Y NY Y Y 2 N N N N N 3 Y Y N Y Y 4 N Y N N Y 5 N N N N Y 6 N Y Y N N 7 N NY N Y 8 Y N Y N N 9 N N Y Y N 10 Y N N N Y 11 Y N N Y N 12 Y N Y Y N 13Y N N N N 14 N Y Y Y Y 15 Y N N Y N 16 Y Y N N Y 17 Y Y N N N 18 Y Y Y YY 19 N Y N Y N 20 Y Y Y Y N 21 N N N Y Y Note: Basal medium (T0 medium)contained 100 g/l glucose, 12.5 g/l KH₂PO₄, 1.0 g/l Na₂HPO₄, 5.0 g/l(NH4)₂SO₄, 2 g/l urea, 2.5 g/l MgSO₄•7H2O, 0.25 g/l CaCl₂•2H2O, 2.4 mg/lCoCl₂, 0.3 mg/l HBO₃ and 1.2 mg/l NaMoO₄, pH 5.5. 0.6 ml of traceelement mix was added to each litre of medium. Where indicated by N, thetrace element was omitted in the trace element mix. The final mediumcontained various combinations of FeSO₄7H₂O (24 mg/l), MnSO₄ (6 mg/l),ZnSO₄ (1.2 mg/l) and CuCl2 (0.6 mg/l).

TABLE 7 Anova Analysis of Table 6 Sum of Mean F p-value Source Squaresdf Square Value Prob > F Remarks Model 469.03 15.00 31.27 17.67 0.0025significant A-FeSO₄ 6.58 1.00 6.58 3.72 0.1118 B-MnSO₄ 5.67 1.00 5.673.20 0.1335 C-ZnSO₄ 305.29 1.00 305.29 172.50 <0.0001 significantD-CUCl₂ 22.61 1.00 22.61 12.78 0.0160 significant E-AlCl₃ 3.46 1.00 3.461.95 0.2211 AB 0.87 1.00 0.87 0.49 0.5151 AC 69.62 1.00 69.62 39.340.0015 significant AD 22.14 1.00 22.14 12.51 0.0166 significant AE 16.671.00 16.67 9.42 0.0278 significant BC 2.88 1.00 2.88 1.63 0.2579 BD 0.441.00 0.44 0.25 0.6408 BE 5.87 1.00 5.87 3.32 0.1282 CD 0.18 1.00 0.180.10 0.7598 CE 0.29 1.00 0.29 0.16 0.7043 DE 19.93 1.00 19.93 11.260.0202 significant Residual 8.85 5.00 1.77 Cor Total 477.88 20.00 Std.Dev. 1.33 R-Squared 0.98 Mean 21.66 Adj R- 0.93 Squared C.V. % 6.14 PredR- 0.29 Squared PRESS 339.46 Adeq 16.34 Precision

In another experiment, the effects of the remaining trace elements,i.e., CoCl₂, HBO₃ and NaMoO₄ were tested. Results showed that CoCl₂ (2.4mg/1) strongly inhibited massoia lactone production (p<0.0001) whileNaMoO₄ (1.2 mg/1) significantly improved the production (p<0.05). HBO₃(0.3 mg/1) did not appear to have significant effect (Tables 8 and 9).The best medium was Run 12, with a massoia lactone yield of 1300 mg/l(FIG. 7B). Notably, this medium contains high level of CaCl₂ (0.25 g/lCaCl₂.2H₂O).

TABLE 8 Trace Elements Part 2 Run # CoCl₂ NaMoO₄ H3BO₄ 1 N Y Y 2 Y Y N 3N N N 4 N Y N 5 N N N 6 Y N Y 7 N Y Y 8 N N Y 9 Y Y Y 10 Y N N 11 N N Y12 N Y N 13 Y Y Y Note: Media were inoculated to 0.1 OD₆₀₀ withwater-washed cell stock cultured in HMDC medium and cultured at 30° C.with 200 rpm shaking for 8 days. Basal medium was T0 medium (Table 6)with various combinations of CoCl₂ (2.4 mg/l), HBO₃ (0.3 mg/l) andNaMoO₄ (1.2 mg/l).

TABLE 9 Anova Analysis of Table 8 Sum of Mean F p-value Source Squaresdf Square Value Prob > F Remarks Model 2702668.48 6.00 450444.75 47.99<0.0001 significant A-CoCl₂ 2431273.40 1.00 2431273.40 259.01 <0.0001significant B-NaMoO₄ 60353.96 1.00 60353.96 6.43 0.0443 significantC-H₃BO₄ 769.68 1.00 769.68 0.08 0.7842 AB 21411.70 1.00 21411.70 2.280.1817 AC 320.50 1.00 320.50 0.03 0.8595 BC 18462.23 1.00 18462.23 1.970.2103 Residual 56320.37 6.00 9386.73 Lack of Fit 3671.82 1.00 3671.820.35 0.5805 not significant Pure Error 52648.56 5.00 10529.71 Cor Total2758988.86 12.00 Std. Dev. 96.89 R- 0.98 Squared Mean 741.48 Adj R- 0.96Squared C.V. % 13.07 Pred R- 0.91 Squared PRESS 243152.35 Adeq 15.75Precision

Example 5 Utilization of Various Carbon Sources by A. melanogenum W5-2

To see if strain W5-2 is able to utilize other carbon sources for theproduction of massoia lactone, glucose in T2 medium was replaced withthe same concentrations of D-(+)-mannose, D-(+)-xylose and glycerol.Mannose was essentially as efficiently utilized as glucose. The strainalso efficiently converted xylose to massoia lactone although the yieldwas about 20% lower than with glucose under the conditions tested.Glycerol was a poor carbon source (FIG. 8).

Example 6 Fed-Batch Fermentation

To verify the performance of the optimized media, fed-batch fermentationwas performed in 2 L bioreactors using T0, T2 and T3 media. As theoriginal A-21M medium barely supports the growth of W-52, a modifiedA-21M medium (MA-21M) was used as a reference. MA-21M contained 120 g/lglucose, 1.5 g/l NaNO₃, 1.0 g/l KNO₃, 0.05 g/l KH₂PO₄, 0.2 g/l MgSO₄,0.0056 g/l FeSO₄, 0.2 g/l Yeast Extract (pH 5.5). Indeed, both T2 and T3media showed drastically improved massoia lactone production compared tothe T0 medium (FIG. 9A). Best result was observed with T3 medium, inwhich massoia lactone level peaked at Day 7, reaching 10.268 g/l, with avolumetric productivity of 61.11 mg/hr/l. In T2 medium, massoia lactoneproduction was significantly lower than T3 medium throughout the timecourse. Maximal production was delayed at least 3 days with a titre of6.924 g/l and volumetric productivity of 28.85 mg/hr/l. In contrast,maximal titre for T0 medium was only 0.441 g/l observed at Day 10, witha volumetric productivity of 1.84 mg/hr/l. The MA-21M medium performedbetter than T0 medium, peaking Day 9 with a titre of 1.777 g/l andvolumetric productivity of 8.23 mg/hr/l. Thus, the volumetricproductivity in T3 and T2 medium were 7.43-fold and 3.51-fold higherthan that of A-21M medium, respectively.

In T0 and T2 medium, cells produced much higher biomass at the cost ofthe desired metabolite (FIG. 9B). Nitrogen source in both T2 and T3media were rapidly consumed and became depleted after Day 2. Incontrast, NH⁴⁺ level remained high in T0 medium until Day 8 (FIG. 9C).The cells showed very different colors: cells contained strong blackpigments, presumably melanin, in T2 and T3 media. In stark contrast withprevious report [6], the result suggests that the production of blackpigment was associated with high massoia lactone production.

Example 7 Effect of Medium pH

W5-2 cells cultured in YPD medium was cultured in T2 medium adjusted tovarious pH values ranging from pH 4 to pH 6.5. The maximal titre wasobserved with pH6.0. Slight reduction of yield was seen for pH 5.5 and6.5 (FIG. 11).

Example 8 Purity of Massoia Lactone

Fed-batch fermentation was performed in 2 L bioreactors using T3 media.The total fermentation broth was hydrolyzed with H₂SO₄. GCMS analysis ofthe showed the high production of a single peak of massoia lactone(FIGS. 12A and 12B). This is in contrast to earlier work withAureobasidium pullalan A-21M, which produced 3-hydroxyldelta-decalactone at ratio of about 1:1.7 [6].

Example 9 Comparison of House-Keeping Genes

The coding sequences (CDS) of five house-keeping genes of W5-2 strainwere identified using known protein sequences of Ustilago maydis as thequery to search against the W5-2 whole transcriptome database. SEQ IDNOs:2, 7, 8, 9 and 10 are the CDS sequence for GPD1, Actin, TEF1A,Tubulin1 and RPB1 (RNA polymerase 2, the largest subunit), respectively.There were high variations in the levels of sequence identity tohomologs of different Aureobasidium species (Tables 10-14). Note that A.pullulans AY4 has been re-classified as A. melanogenum AY4. For example,GDP1 genomic sequence shares at least 99% identity to homologs of A.melanogenum species at least over 98% of SEQ ID NO:2 while theinter-species homology for GPD1 CDS is below 97% over 98% of SEQ ID NO:2(Table 10). Similarly, TEFA genomic sequence shares at least 99%identity to homologs of A. melanogenum species over 99% of SEQ ID NO:8while the inter-species homology for TEF1A CDS is below 98% over 94% ofSEQ ID NO:8 (Table 12). RPB1 (SEQ ID NO:10) sequence is the mostdivergent.

RPB1 genomic sequence shares at least 96% identity to homologs of A.melanogenum species over 98% of SEQ ID NO:10 while the inter-specieshomology for RPB1 CDS is below 90% over 92% of SEQ ID NO:10 (Table 14).

TABLE 10 BLASTn Search Results of GPD1 (SEQ ID NO: 2) Max Total Query EDescription score score cover value Ident Accession Aureobasidiummelanogenum CBS 1459 1812 98% 0.0 99% AYEN01000011.1 110374scaffold_4_c3, whole genome shotgun sequence Aureobasidium pullulans AY41448 1795 98% 0.0 99% AMCU01000020.1 contig20, whole genome shotgunsequence Aureobasidium namibiae CBS 1371 1692 98% 0.0 97% AYEM01000004.1147.97 scaffold_2_c1, whole genome shotgun sequence Aureobasidiumpullulans isolate 1321 1635 98% 0.0 96% LVWM01000514.1 Santandercontig_514, whole genome shotgun sequence Aureobasidium subglaciale EXF-1315 1636 98% 0.0 96% AYYB01000004.1 2481 scaffold_ 2_c1, whole genomeshotgun sequence Aureobasidium pullulans EXF-150 1293 1605 98% 0.0 95%AYEO01000008.1 scaffold_6_c1, whole genome shotgun sequence

TABLE 11 BLASTn Search Results of Actin (SEQ ID NO: 7) Max Total Query EDescription score score cover value Ident Accession Aureobasidiumpullulans 1216 2960 99% 0.0 92% AMCU01000120.1 AY4 contig120, wholegenome shotgun sequence Aureobasidium melanogenum 1210 2960 99% 0.0 92%AYEN01000004.1 CBS 110374 scaffold_3_c1, whole genome shotgun sequenceAureobasidium namibiae 917 1016 44% 0.0 86% AYEM01000024.1 CBS 147.97scaffold_18_c1, whole genome shotgun sequence Aureobasidium pullulans828 1993 96% 0.0 84% LVWM01000225.1 isolate Santander contig_225, wholegenome shotgun sequence Aureobasidium pullulans 811 1872 93% 0.0 84%AYEO01000002.1 EXF-150 scaffold_2_c1, whole genome shotgun sequenceAureobasidium subglaciale 614 735 39% 4e−174 82% AYYB01000022.1 EXF-2481scaffold_19_c1, whole genome shotgun sequence

TABLE 12 BLASTn Search Results of TEF1A (SEQ ID NO: 8) Max Total Query EDescription score score cover value Ident Accession Aureobasidiummelanogenum CBS 2041 2472 99% 0.0 99% AYEN01000021.1 110374scaffold_11_c1, whole genome shotgun sequence Aureobasidium pullulansAY4 2041 2472 99% 0.0 99% AMCU01000064.1 contig64, whole genome shotgunsequence Aureobasidium namibiae CBS 2015 2368 94% 0.0 98% AYEM01000007.1147.97 scaffold_4_c1, whole genome shotgun sequence Aureobasidiumpullulans EXF-150 1881 2112 92% 0.0 96% AYEO01000027.1 scaffold_17_c1,whole genome shotgun sequence Aureobasidium subglaciale EXF- 1753 198593% 0.0 94% AYYB01000027.1 2481 scaffold_23_c1, whole genome shotgunsequence

TABLE 13 BLASTn Search Results of Tubulin1 (SEQ ID NO: 9) Max TotalQuery E Description score score cover value Ident AccessionAureobasidium melanogenum CBS 110374 1284 2316 97% 0.0 98%AYEN01000040.1 scaffold_23_c2, whole genome shotgun sequenceAureobasidium pullulans AY4 contig32, 1284 2305 97% 0.0 98%AMCU01000032.1 whole genome shotgun sequence Aureobasidium namibiae CBS147.97 1194 2187 97% 0.0 96% AYEM01000023.1 scaffold_ 17_c1, wholegenome shotgun sequence Aureobasidium pullulans isolate Santander 11601981 92% 0.0 95% LVWM01000153.1 contig_153, whole genome shotgunsequence Aureobasidium pullulans EXF-150 1158 1984 92% 0.0 95%AYEO01000003.1 scaffold_3_c1, whole genome shotgun sequenceAureobasidium subglaciale EXF-2481 1140 2033 97% 0.0 95% AYYB01000001.1scaffold_0_c1, whole genome shotgun sequence

TABLE 14 BLASTn Search Results of RPB1 (SEQ ID NO: 10) Max Total Query EDescription score score cover value Ident Accession Aureobasidiummelanogenum CBS 110374 7915 9706 98% 0.0 96% AYEN01000002.1scaffold_1_c2, whole genome shotgun sequence Aureobasidium pullulans AY4contig81, 7866 9631 99% 0.0 96% AMCU01000081.1 whole genome shotgunsequence Aureobasidium namibiae CBS 147.97 6032 6855 91% 0.0 89%AYEM01000002.1 scaffold_1_c2, whole genome shotgun sequenceAureobasidium subglaciale EXF-2481 5389 6038 91% 0.0 87% AYYB01000017.1scaffold_15_c1, whole genome shotgun sequence Aureobasidium pullulanscontig_319, 5334 5999 92% 0.0 86% LVWM01000319.1 whole genome shotgunsequence Aureobasidium pullulans contig_233, 5334 5999 92% 0.0 86%LVWM01000233.1 whole genome shotgun sequence Aureobasidium pullulanscontig_45, whole 5334 5999 92% 0.0 86% LVWM01000045.1 genome shotgunsequence Aureobasidium pullulans EXF-150 5323 5993 92% 0.0 86%AYEO01000028.1 scaffold_18_c1, whole genome shotgun sequence

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

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1. A pure culture of Aureobasidium melanogenum that expresses nofunctional Aureobasidin A biosynthesis complex gene mRNA when cultured.2. The pure culture of claim 1, wherein the Aureobasidium melanogenumstrain is Aureobasidium melanogenum strain W5-2.
 3. The pure culture ofclaim 2, wherein Aureobasidium melanogenum W5-2 is deposited withAgricultural Research Culture Collection (NRRL) and assigned AccessionNumber NRRL
 67063. 4. The pure culture of claim 1, wherein theAureobasidium melanogenum GDP1 sequence shares at least 97.5% identityover at least 98.5% of SEQ ID NO:2, preferably 99%-100% identity to overat least 98% of SEQ ID NO:2.
 5. The pure culture of claim 1, wherein theAureobasidium melanogenum TEF1A sequence shares at least 98% identityover at least 94% of SEQ ID NO:8, preferably 99%-100% identity to overat least 99% of SEQ ID NO:8.
 6. The pure culture of claim 1, wherein theAureobasidium melanogenum RBP1 sequence shares at least 91% identityover at least 92% of SEQ ID NO:10, preferably 96%-100% identity to overat least 98% of SEQ ID NO:10.
 7. The pure culture of claim 1, whereinthe Aureobasidium melanogenum GDP1 sequence shares at least 97.5%identity over at least 98.5% of SEQ ID NO:2, preferably 99%-100%identity to over at least 98% of SEQ ID NO:2, wherein the Aureobasidiummelanogenum TEF1A sequence shares at least 98% identity over at least94% of SEQ ID NO:8, preferably 99%-100% identity to over at least 99% ofSEQ ID NO:8 and wherein the Aureobasidium melanogenum RBP1 sequenceshares at least 91% identity over at least 92% of SEQ ID NO:10,preferably 96%-100% identity to over at least 98% of SEQ ID NO:10. 8.Use of the pure culture of claim 1 for the fermentative production ofmassoia lactone.
 9. A method of fermentative production of massoialactone comprising culturing an Aureobasidium melanogenum species in aculture medium comprising high levels of phosphate ions, ammonium ions,calcium ions, at least two trace elements selected from the groupconsisting of Fe²⁺, Cu²⁺, Zn²⁺ and MoO₄ ²⁻, urea and a carbon sourceselected from the group consisting of glucose, mannose, xylose andmixtures thereof for a sufficient period of time to produce massoialactone in a fermentation product.
 10. The method of claim 9, whereinthe pH of the culture medium is from about 5.5 to about 6.5, preferably6.0
 11. The method of claim 9, wherein the amount of urea present in theculture medium is from about 1.5 g/l to about 2.5 g/l, preferably fromabout 1.8 g/l to about 2.2 g/l, more preferably about 2 g/l.
 12. Themethod of claim 9, wherein the carbon source present in the culturemedium is from about 4% to about 12%, preferably from about 5% to about12%, preferably from about 5% to about 11%, more preferably from about5% to about 10%.
 13. The method of claim 9, wherein each trace elementin the culture medium is present in an amount from about 0.1 μM to about1.0 mM, from about 1.0 μM to about 1.0 mM, from about 10.0 μM to about1.0 mM, or from about 100 μM to about 1.0 mM.
 14. The method of claim 9,wherein the culture comprises about 10.0 g/l to about 15 g/l, preferablyabout 12.5 g/l KH₂PO₄, about 0.5 g/l to about 2.0 g/l, preferably about1.0 g/l Na₂HPO₄, about 3.5 g/l to about 6.5 g/l, preferably about 5.0g/l (NH4)₂SO₄, about 1.0 g/l to about 4.0 g/l, preferably about 2.5 g/lMgSO₄.7H₂O and about 0.10 g/l to about 0.40 g/l, preferably about 0.25g/l CaCl₂.2H₂O.
 15. The method of claim 14, wherein the culture mediumfurther comprises about 2 g/l urea, about 5% to about 10% carbon sourceand at least two of the trace elements.
 16. The method of claim 9,wherein the period of time is for about 4 days to about 12 days,preferably for about 5 days to about 12 days, preferably for about 5days to about 11 days, preferably for about 6 days to about 11 days,more preferably for about 7 days to about 10 days.
 17. The method ofclaim 9, wherein the Aureobasidium melanogenum is a strain thatexpresses no functional Aureobasidin A synthase gene mRNA in culture.18. The method of claim 17, wherein the Aureobasidium melanogenum strainis Aureobasidium melanogenum W5-2.
 19. The method of claim 18, whereinAureobasidium melanogenum W5-2 is deposited with Agricultural ResearchCulture Collection (NRRL) and assigned Accession Number NRRL
 67063. 20.The method of claim 9, which further comprises hydrolyzing thefermentation product by a strong inorganic acid and purifying themassoia lactone from the fermentation product.
 21. The method of claim9, wherein the fermentation product is substantially free of contaminant3-hydroxyl delta-decalactone (3-hydroxydecan-5-olide).
 22. A culturemedium for the fermentative production of massoia lactone byAureobasidium melanogenum species, the culture medium comprising highlevels of phosphate ions, ammonium ions, calcium ions, at least twotrace elements selected from the group consisting of Fe²⁺, Cu²⁺, Zn²⁺and MoO₄ ²⁻, urea and a carbon source selected from the group consistingof glucose, mannose, xylose and mixtures thereof.
 23. The culture mediumof claim 22, wherein the pH of the culture medium is from about 5.5 toabout 6.5, preferably 6.0
 24. The culture medium of claim 22, whereinthe amount of urea present in the culture medium is from about 1.5 g/lto about 2.5 g/l, preferably from about 1.8 g/l to about 2.2 g/l, morepreferably about 2 g/l.
 25. The culture medium of claim 22, wherein thecarbon source present in the culture medium is from about 4% to about12%, preferably from about 5% to about 12%, preferably from about 5% toabout 11%, more preferably from about 5% to about 10%.
 26. The culturemedium of claim 22, wherein each trace element present in the culturemedium is present in an amount from about 0.1 μM to about 1.0 mM, fromabout 1.0 μM to about 1.0 mM, from about 10.0 μM to about 1.0 mM, orfrom about 100 μM to about 1.0 mM.
 27. The culture medium of claim 22,wherein the culture medium comprises about 10.0 g/l to about 15 g/l,preferably about 12.5 g/l KH₂PO₄, about 0.5 g/l to about 2.0 g/l,preferably about 1.0 g/l Na₂HPO₄, about 3.5 g/l to about 6.5 g/l,preferably about 5.0 g/l (NH4)₂SO₄, about 1.0 g/l to about 4.0 g/l,preferably about 2.5 g/l MgSO₄.7H₂O and about 0.10 g/l to about 0.40g/l, preferably about 0.25 g/l CaCl₂.2H₂O.
 28. The culture medium ofclaim 27, wherein the culture medium further comprises about 2 g/l urea,about 5% to about 10% carbon source and at least two of the traceelements.
 29. The culture medium of claim 22, wherein the culture mediumfurther comprises an Aureobasidium melanogenum species.
 30. The culturemedium of claim 29, wherein the Aureobasidium melanogenum is a strainthat expresses no functional Aureobasidin A synthase gene mRNA inculture.
 31. The culture medium of claim 30, wherein the Aureobasidiummelanogenum strain is Aureobasidium melanogenum W5-2.
 32. The culturemedium of claim 31, wherein Aureobasidium melanogenum W5-2 is depositedwith Agricultural Research Culture Collection (NRRL) and assignedAccession Number NRRL
 67063. 33. The culture medium of claim 22 furthercomprising more than 10 g/l of massoia lactone.